The substantia nigra modulates proximal colon tone and motility in a vagally-dependent manner in the rat

Abstract

A monosynaptic pathway connects the substantia nigra pars compacta (SNpc) to neurons of the dorsal motor nucleus of the vagus (DMV). This monosynaptic pathway modulates the vagal control of gastric motility. It is not known, however, whether this nigro-vagal pathway also modulates the tone and motility of the proximal colon. In rats, microinjection of retrograde tracers in the proximal colon and of anterograde tracers in SNpc showed that bilaterally labelled colonic-projecting neurons in the DMV received inputs from SNpc neurons. Microinjections of the ionotropic glutamate receptor agonist, NMDA, in the SNpc increased proximal colonic motility and tone, as measured via a strain gauge aligned with the colonic circular smooth muscle; the motility increase was inhibited by acute subdiaphragmatic vagotomy. Upon transfection of SNpc with pAAV-hSyn-hM3D(Gq)-mCherry, chemogenetic activation of nigro-vagal nerve terminals by brainstem application of clozapine-N-oxide increased the firing rate of DMV neurons and proximal colon motility; both responses were abolished by brainstem pretreatment with the dopaminergic D1-like antagonist SCH23390. Chemogenetic inhibition of nigro-vagal nerve terminals following SNpc transfection with pAAV-hSyn-hM4D(Gi)-mCherry decreased the firing rate of DMV neurons and inhibited proximal colon motility. These data suggest that a nigro-vagal pathway modulates activity of the proximal colon motility tonically via a discrete dopaminergic synapse in a manner dependent on vagal efferent nerve activity. Impairment of this nigro-vagal pathway may contribute to the severely reduced colonic transit and prominent constipation observed in both patients and animal models of parkinsonism. KEY POINTS: Substantia nigra pars compacta (SNpc) neurons are connected to the dorsal motor nucleus of the vagus (DMV) neurons via a presumed direct pathway. Brainstem neurons in the lateral DMV innervate the proximal colon. Colonic-projecting DMV neurons receive inputs from neurons of the SNpc. The nigro-vagal pathway modulates tone and motility of the proximal colon via D1-like receptors in the DMV. The present study provides the mechanistic basis for explaining how SNpc alterations may lead to a high rate of constipation in patients with Parkinson's Disease.

Keywords: brain stem; dorsal motor nucleus of the vagus; proximal colon motility; substantia nigra pars compacta.

Figure 1.. SNpc provides an excitatory input to neurons of the dorsal motor nucleus of the vagus that innervate the proximal colon.

A. Representative micrograph of vagal efferent fibers apposing myenteric neurons of the proximal colon. Following microinjection of an anterograde tracer (magenta) in the dorsal vagal complex (DVC; N=4), labeled fibers are observed in PGP9.5-IR myenteric neurons of the proximal colon (magenta arrows). Some PGP9.5-IR neurons appeared to receive close contacts from vagal efferent fibers and were encircled with dextran labeled fibers (white arrows). B. Representative micrograph of neurons in the DMV. Following microinjection of an anterograde tracer in the SNpc, labeled fibers (white arrows) can be seen apposing DMV neurons labelled following injections of the retrograde tracer CTB in the proximal colon (N=5). C. Representative micrographs of ChAT-IR neurons (brown) and c-fos (blue-black) within the within the DMV. Black arrows indicate c-fos-IR neurons within the lateral DMV following NMDA microinjection into the DMV. Scale bars: 100μm in all panels. DMV: dorsal motor nucleus of the vagus; NTS: nucleus tractus solitarius; 4V: fourth ventricle

Figure 2.. NMDA microinjection in the SNpc increases the tone and motility of the proximal colon.

A. Representative micrograph showing the location of a NMDA injection (arrow) in SNpc (Aa, scale bar: 500μm). Schematic image summarizing the location of the injections in SNpc (Ab, N=21). B. Representative trace showing that the NMDA (5nmoles/210nl) microinjection in the left SNpc (arrow) increases both tone and motility of the proximal colon. C. Summary graphic showing the increase in motility (N=21, p<0.0001, one tailed paired t test) and tone (N=21) of the proximal colon upon NMDA microinjection in the left SNpc. D. Representative trace showing that the AMPA (5nmoles/210nl) microinjection in the left SNpc (arrow) increases both tone and motility of the proximal colon. E. Summary graphic showing the increase in motility (N=4, p=0.0463, one tailed paired t test) and tone (N=4) of the proximal colon upon AMPA microinjection in the left SNpc.

Figure 3.. Vagotomy prevents the increase in tone and motility of the proximal colon following SNpc microinjection of NMDA microinjection.

A. Representative traces showing that the increase in tone and motility of the proximal colon induced by NMDA (5nmoles/210nl, arrow) microinjection in the left SNpc (upper panel) is prevented by vagotomy (lower panel). B. Summary graphic showing the increase in motility and tone observed in response to NMDA before (N=5; p=0.0097 NMDA vs baseline, one tailed paired t test) and after vagotomy (ns: p=0.3263 Vagotomy+NMDA vs baseline, two tailed paired t test; tone: p=0.0097 NMDA vs. Vagotomy +NMDA, one-tailed paired t-test). ns: not significant.

Figure 4.. Chemogenetic activation of nigro-vagal projections increases the tone and motility of the proximal colon.

A. Schematic diagram illustrating the experimental protocol used for chemogenetic manipulation of the nigro-vagal pathway. B. Representative micrograph showing the location of hM3D(Gq) (upper left; red) and empty vector (upper right, yellow) microinjection. SNpc neurons are labeled with tyrosine hydroxylase. Representative micrograph showing the location of the mCherry and EYFP labelled fibers in the DMV following SNpc transfection, and apposing choline acetyl transferase-IR neurons (lower panels). C. Recording of proximal colon function in an empty vector transfected rat following application of CNO to the 4^th ventricle. D. Graphical summary of the change in proximal colon motility (left, N=6; p=0.5114, two tailed paired t test) and tone (right) in empty vector transfected rats in response to brainstem application of CNO. E. Recording of proximal colon function in an hM3D(Gq) transfected rat following application of CNO to the 4^th ventricle. F. Graphical summary of the change in proximal colon motility (left, N=7; p=0.0434, two tailed paired t test) and tone (right) in hM3D(Gq) transfected rats following brainstem application of CNO. G. Recording of proximal colon function in an hM4D(Gi) transfected rat following application of CNO to the 4^th ventricle. H. Graphical summary of the change in proximal colon motility (left, N=5; p=0.0051, two tailed paired t test) and tone (right) in hM4D(gi) transfected rats following brainstem application of CNO. Scale bars: 100μm; ns: not significant.

Figure 5.. Chemogenetic activation of nigro-vagal projections modulates DMV neuronal activity

Whole cell patch clamp recordings from a DMV neuron current clamped at a potential to allow spontaneous firing at approximately 1 event/sec. A. In an empty vector transfected rat, superfusion with CNO had no effect on action potential firing rate. B. Graphical summary of the effects of CNO on action potential firing rate in DMV neurons from empty vector transfected rats (N=7, p=0.5300; two-tailed paired t-test). C. In an hM3D(Gq) transfected rat, superfusion with CNO increased action potential firing in a manner that was reversed following superfusion with the D1-like receptor antagonist, SCH23390. D. Graphical summary of the effects of CNO to alter action potential firing rate in DMV neurons from hM3D(Gq) transfected rats (N=7; p=0.0454 baseline vs CNO, p=0.0112 CNO vs SCH, p=0.4204 baseline vs SCH, one-way ANOVA, mixed effects model). E. In an hM4D(Gi) transfected rat, superfusion with CNO decreased action potential firing in a reversible manner. F. Graphical summary of the effects of CNO to alter action potential firing rate in DMV neurons from hM4D(Gq) transfected rats (N=7; p=0.0257; two-tailed paired t-test). Bars indicate a 2min interval. ns: not significant.

Figure 6.. Pretreatment with the dopamine D1-like antagonist SCH23390 prevents the increase in tone and motility of the proximal colon following NMDA microinjection in the left SNpc.

A. Representative traces showing that the increase in tone and motility of the proximal colon induced by NMDA (5nmoles/210nl, arrow) microinjection in the left SNpc (A, left) is prevented by 4^th ventricular application of the dopamine D1-like antagonist SCH23,390 (45nmoles/2μl) (A, right). B. Summary graphic showing that the increase in motility (N=4; p=0.0204, one-tailed paired t-test) and tone (N=5) of the proximal colon observed in response to SNpc microinjection of NMDA is prevented by pretreatment with SCH23390 in the IV ventricle (motility: p=0.3974 vs baseline, one-tailed paired t-test; tone: p=0.0266, one-tailed paired t test). 4th ventricular application of SCH23390 had no effect on motility (N=5; p=0.1491 vs baseline, two-tailed paired t test) or tone of the proximal colon. C. Representative traces showing that the increase in tone and motility of the proximal colon induced by NMDA (5nmoles/210nl, arrow) microinjection in the left SNpc (left) is not affected by pretreatment with the dopamine D2-like antagonist L741646 (45nmoles/2μl) on the floor of the IV ventricle (right). D. Summary graphic showing that the NMDA-induced increase in motility (N=5; p=0.0018, one-tailed paired t-test) and tone (N=5) of the proximal colon is not altered by pretreatment with L741646 (motility: p=0.3996 NMDA vs L741646+NMDA, two-tailed paired t test; tone p=0.8051, two-tailed paired t test). 4th ventricular application of the D2-like antagonist L741646 increased both the motility (N=5; p=0.0088 vs baseline, one-tailed paired t test) and the tone. Bars indicate a 40min interval. ns: not significant.

Figure 7.. Pretreatment with the dopamine D2-like antagonist L741646 attenuates the decrease in motility of the proximal colon following DA microinjection in the DVC.

A. Representative micrograph showing the location of a dopamine (DA) injection (arrow) in the DMV (Aa, scale bar: 100μm). Schematic image summarizing the location of the injection of DA in the DMV (Ab, N=5, other microinjection locations were omitted for clarity). DMV: dorsal motor nucleus of the vagus; NTS: nucleus tractus solitarius; cc: central canal; AP area postrema B. Representative trace showing that DVC microinjection of DA (100nmoles/60nl; arrow) decreases both the tone and motility of the proximal colon. C. Summary graphic showing the decrease in motility (left; N=10; p<0.0001, one-tailed paired t test) and tone (right; N=10) of the proximal colon upon DA microinjection in the left DMV. D. Representative traces showing that the decrease in motility and tone of the proximal colon induced by DVC microinjection of DA (100nmoles/60nl; left trace) attenuated by 4^th ventricular application of the dopamine D2-like antagonist L741646 (45nmoles/2μl; right trace). Bars indicate a 40min.interval. E. Summary graphic showing that the decrease in proximal colon motility (N=5; p=0.01, one-tailed paired t-test) and tone (N=4) observed after DVC microinjection of DA is attenuated by IV ventricle application of L741646 (motility: p=0.2386 baseline vs. L741626+DA, two-tailed paired t-test, p=0.0361 DA vs. L741626+DA one-tailed paired t-test; tone: p=0.0414 DA vs. L741626+DA, one-tailed paired t-test). Bars indicate a 40min interval. ns: not significant.